Limb devlopment

M.Sc. 2012

Human embryo -32 days

Placodes sensory placodes, lens pit, otocyst, nasal placode, primary/secondary vesicles, fourth ventricle of brain Mesoderm continued segmentation of paraxial mesoderm (somite pairs), heart prominence Head 1st, 2nd and 3rd pharyngeal arch, forebrain, site of lens placode, site of otic placode, stomodeum Body - heart, liver, umbilical cord, mesonephric ridge visible externally as bulges. Limb upper and lower limb buds growing Neural first appearance of the future cerebral hemispheres. Cerebellar plate differentiated to an intermediate layer, and future rhombic lip identifiable Ventricular System Subarachnoid space initially as irregular spaces on the ventral surface of the spinal cord. Liver hepatic gland and its vascular channels enlarge, hematopoietic function appears Eye - Lens the lens placode is indented by the lens pit

LIMB DEVELOPMENT
Each limb results from a developmental field. The developmental fields are determined during gastrulation. These limb fields are established by the expression of HOX genes. The expression of Tbx-4 causes the limb to develop into a forelimb and expression of the factor Tbx-5 causes the limb to develop into a hind limb. Beginning from the fourth week from fertilization, over a period of 25 days, a complex of genetic signals control the intricate pathways that result in a limb with the correct orientation, size, and number of digits. Limb development is a continuous process divided into four stages: 1. bud stage (initial outgrowth), 2. paddle stage (dorsoventral flattening), 3. the plate stage (relative expansion of the distal end), 4. rotation stage (rotation around the proximodistal axis).

1. LIMB BUD FORMATION

34 day old human embryo (5mm) 34 pairs of somites Forelimb (lower left) started to develop Hindlimb just beginning (right side) By day 37,In upper limb bud : 1. nerves: median nerve, radial nerve and ulnar nerve entered into hand plate, 2. myoblasts: spindle shaped and oriented parallel to limb bud axis

Limb Bud formation(contd.)

The limbs of the embryo develop from buds that protrude from the side of the main body axis. Limb buds arise on the lateral body at the level of sclerotomes as ectoderm and mesoderm (somite) proliferations. Each limb bud consists of a mesenchymal core of mesoderm covered by an ectodermic cap. Limb buds will become the early arms and legs of the embryo. The upper limbs appear before the lower limbs that are delayed about two days in respect to the upper limbs. At the early stages of embryonic development, the forelimb and hind limb buds look like paddles on either side of the embryo and are indistinguishable from one another.

2. LIMB PADDLE FORMATION

The limb buds continue their formation by the migration and proliferation of the differentiating mesenchymal tissues. The ectoderm at the tip of the bud thickens to form a specialized structure, called the apical ectodermal ridge. This structure is the signaling center that allows proper growth along the proximodistal axis (shoulders to digits). Along with it , the limb becomes flattened along the dorsoventral axis (back of hand to palm) and asymmetric along the anteroposterior axis (thumb to little finger). Proximodistal, dorsoventral, and anteroposterior axes represent the routes of the normal limb growth. The most proximal structure (stylopod) begins to differentiate first, followed by the progressive differentiation of more distal structures (zeugopod and autopod).

Limb paddle (contd.)

This outgrowth and patterning depends on the establishment and maintenance of other signaling centers within the limb bud, named the zone of polarizing activity located in the mesenchyme at the posterior margin of the bud, and the non ridge ectoderm of the bud. These developmental components are interdependent and, through a series of reciprocal signals and feedback systems, yield the correct tissue patterning and growth. Each bud develops to form a complex of interconnected limb elements comprised of bone, muscle, and tendon characteristic of either the fore limb or hind limb. The actual trigger for limb bud initiation is still unknown, although likely candidates have been identified as Fibroblast Growth Factor 8 and 10.

3.PLATE STAGE
The plate stage is characterized by the formation of flattened platelike areas on the distal ends of the limbs called the hand plates and foot plates They are flattened along the dorsoventral axis. Within these distal plates, some structure is noticeable. There are radially arranged thickenings called digital rays (precursors of the digits). Between the digital rays are thin areas where cells begin to undergo apoptosis (programmed cell death) that allow the separating of the digits. The thin areas are called interdigital grooves. This arrangement gives rise to free digits. A constriction on the limb just proximal to the hand and footplates, called primary constrictions of the limb, is evident in this stage. These constrictions will develop into wrists and ankles. At approximately seven weeks, the longitudinal axes of the upper and lower limb buds are parallel.

4. ROTATION
In the rotation stage, the position of the limb buds relative to the trunk change in a predetermined manner not related to muscle activity or inherent osseous torsion. During this stage, the rotation of the limbs creates a three dimensional structure. Because of the differential growth of the cartilage model that continue to elongate the limb, different parts grow at different rates. This causes a twisting or rotation of each limb around its proximodistal axis. Upper limbs rotate one way (laterally or externally), while lower limbs rotate the other way (medially or internally) bringing the great toe to the midline from its initial postaxial position. This creates the characteristic positions of the limbs with the point of the elbow facing caudally and dorsally, and the knee facing cranially and ventrally. Consequently, the equivalent bones and muscles of the upper and lower limbs are oriented 180 degrees apart. This means that in the structural organization of the upper and lower limbs, their flexors and extensors are positioned on opposite sides and the movements at equivalent joints are in opposite directions

Carnegie stage 12 to 23 Human Forelimb Development:

Summary
In the 5th week hand and footplates appear at the ends of limb buds and ridges form digital rays. Cells between the digital rays are removed by programmed cell death (apoptosis). Late in Carnegie stage embryogenesis (Stage 20-23, 8th week) limb rotation occurs. Forelimbs and hindlimbs rotate in different directions, upper limb rotates dorsally, lower limb rotates ventrally, thumb and toe rostral, knee and elbow face outward. Bones within the limb form by endochondrial ossification (begins Carnegie stage 18) throughout embryo. This process is the replacement of cartilage with bone (week 512).

Timeline limb development

By day 44,Limb Bone forms by endochondrial ossification and throughout embryo replacement of cartilage with bone (week 5-12). By day 50, upper limbs begin to rotate ventrally By day 53, fingers and toes lengthen By day 56, upper limbs longer and bent at elbow, hands and feet turned inward, foot with separated digits, wrist, hand with separated digits By day 64, fingernails appear By week 14,toenails appear

Molecular control of limb formation

Limb bud
Limb bud: Mesoderm & Epithelial Ectoderm Ectoderm over mesoderm Ectoderm thickened as Apical Ectodermal Ridge (AER)

Cells that Contribute to Mesoderm of the Limb Bud

Limb mesoderm (mesenchyme) comes from the somite and lateral plate mesoderm The Lateral Plate Mesoderm contributes to the skeleton, blood vessels & connective tissue The Somite Mesoderm contributes to the Musculature Nerve cells & Neural crest cells migrate in as well Motor Axons from spinal cord will innervate limb Neural Crest gives rise to sensory nerves, schwann cells, pigment cells

 Apical ectodermal ridge (AER). Secretes fibroblast growth factor (FGF) proteins. Required for limb growth and patterning along the proximal-distal axis. Required for pattern formation along the dorsal-ventral axis.

Early limb dvelopment Limb grows & develops proximo-distally Zone of Cell Division: Region of actively dividing cells Zone of Differentiation: Region of cell specialization

 Organizer regions.

Zone of polarizing activity (ZPA).

Secretes Sonic hedgehog, a protein growth factor. Required for pattern formation of the limb along the anterior-posterior axis. Homeobox-containing (Hox) genes play a role in specifying the identity of regions of the limb, as well as the body as a whole.

The Organization and Polarity of the Developing Limb Bud The limb bud has a strict pattern and polarity. Development is organized around the A-P, D-V and P-V axes. The tissues of the limb will differentiate in a specific pattern that is defined in part by the existing embryonic regions: the Apical Ectodermal Ridge (AER), the Zone of Polarizing Activity (ZPA) and the Progress Zone (PZ). The AER acts as the organizing region for the proximodistal axis of the limb. The ZPA organizes the limb along the A-P axis. It does this in part through the expression of Sonic hedgehog resulting in the production of the soluble sonic hedgehog protein. Sonic hedgehog mediates many developmental events. In the limb it not only meditates A-P Axis formation but also the maintenance of the AER.

EPITHELIAL-MESENCHYMAL INTERACTIONS DURING LIMB DEVELOPMENT

Experiments originally done in chickens Modified here to show how results might apply to human limb Removal of AER stops limb development Addition of AER causes formation of 2nd limb Splitting AER leads to 2nd limb

Inferences : AER controls limb development

Limb mesoderm dictates limb development; almost any epithelial ectoderm can replace normal limb epithelium Type of limb depends on type of mesoderm Not species specific: Inter-specific grafts show same induction Inducer may be universal

Pattern Formation in the Vertebrate Limb is a chain of events involving cell signaling and differentiation. . Induction plays a major role in pattern formation. Positional information, supplied by molecular cues, tells a cell where it is relative to the animals body axes

e.g.Limb development in chicks as a model of pattern formation. Wings and legs begin as limb buds. Each component of the limb is oriented with regard to three axes: Proximal-distal Anterior-posterior Dorsal-ventral.

Transcription factor code for developmental identities (particular region)

HOM
Rostral-caudal axis

HOX
limb

Structure and actionconserved

Hox: homeotic selector-fly mutant: transform one part of the body into another Homeodomain-bind to DNA-TF( regulate a large set of downstream genes)

Homeotic transformation of the wing and haltere

Homeotic genesmutated into homeosis transformation Bithorax-haltere into wing

Mutation in HoxD13synpolydactyly
Extra digits & interphalangeal webbing (hetero) Similar but more severe & bony malformation of hands, wrists (Homo)

Transplantation experiment
Organizers in animal embryos: Spemann organizer and ZPA
Spemann organizer

Action of morphogen (paracrine signal)

Hedgehog Drosophila mutant (alter epidermal bristles) Different concentrations to different fates

Human Sonic headehog (notochord secreted to induce brain and spinal cord development) High conc.-neural tube Low conc. motor neurons

In limb (asymmetrical pattern of Digits)-zone of polarizing activity

Signal Transduction & Limb Formation

During limb development the limb bud grows away from the body in a proximo(close) distal (away from) fashion. Developmentally, as the limb bud lengthens and limb components are specified and start to differentiate, what were once distal regions become proximal as new distal regions form. This continues until the limb is fully developed and the final relationship of limb components is defined. For exampe , in early embryogenesis, the humerus as it forms is initially the most distal component but once the radius and ulna and subsequent components form, it becomes proximal to them. PROGRESS-ZONE MODEL OF LIMB DEVELOPMENT, 1. The AER secretes FGF that influences the closest cells (those in the progress zone) to develop into distal structures. FGF is a distalizing factor in limb development. Those cells that are not within range of the AERs influence remain proximal in nature. 2. As the AER extends out due to the continued division of cells in the progress zone, it continues to affect the closest cells by causing them to be specified as distal structure cells. 3. Those that fall out of the range of influence of the AER are no longer influenced by the effects of FGF and retain their previously defined status (i.e., are now proximal components not influenced by the distalizing effect of FGF).

Events of Signal Transduction & Limb Formation

1. Fibroblast Growth Factor (FGF) family of factors is linked to the initiation of bud formation, maintaining bud outgrowth, and the induction of a regeneration 2. FGFs are secreted primarily by AER 3. Tyrosine kinase FGF receptor is expressed on the surface mesenchyme cells 4. FGF Released by AER binds to FGF Receptor (a receptor tyosine kinase or RTK) & activates It 5. RTK then phosphorylates critical proteins 6. This causes the mesenchyme cells to release retinoic acid (RA) 7. RA induces Hox Gene Expression in target cells

FGFs
FGFs produced in the AER serve at least two major functions. 1. to stimulate the proliferation of cells in the progress zone due to their mitogenic activities for limb bud mesenchyme and thus produce the new cells required for limb outgrowth. 2. to maintain Shh expression in the ZPA. FGF 4 although not required to induce Shh expression is largely responsible for maintenance of its expression as the limb elongates. The regulatory interaction between FGF4 and Shh could be reciprocal as Shh produced in the ZPA induces and maintains FGF 4 expression in the AER. This positive feedback loop between FGF 4 and Shh could be one of the mechanisms by which outgrowth and patterning of limb would be coordinately regulated, although additional molecules such as Wnt7a are likely to play a role in regulating Shh expression.

FGFs
One of the target for FGF signaling from the AER is FGF 10 which is expressed in the distal limb bud mesenchyme. This factor is able to interact with FGF 8 and there might be a positive feed-back loop between FGF 10 and FGF 8. This reciprocal regulation is likely to be mediated by two isoforms of FGFR 2, FGFR 2b (that binds FGF 10 exclusively) and FGFR 2c (that binds FGF 8). A recent model has been proposed in which FGF 10 made in the mesenchyme of the limb field diffuses in the ectoderm where it binds FGFR 2b and induces FGF 8 in the ectoderm. The FGF 8 in turn diffuses into the mesoderm and activates FGFR 2c which causes the upregulation of FGF 10. The FGF 10 then continues the loop and results in limb bud induction. Hence FGFR 2 appears to be essential for limb bud initiation whereas FGFR 1 seems to play an essential role at several stages of limb development. This assertion is based on the study of mouse models and expression patterns which have revealed an important function of FGFR 1 in specification of P-D axis formation. FGFR 1-mediated signals are required for maintaining ZPA and progress zone activities.

Thalidomide-induced embryopathy

Proximal-distal axis PZ: progress zone, AER: apical ectodermal ridge, FGF: fibroblast growth factor Teratology-teratogens Thalidomidedamaging tissue within the proliferating center

Role of HOX ,BMP

Some of the FGF in conjonction with Shh can affect expression of the bone morphogenetic protein (Bmp-2 and 7) and Hox genes, mostly Hoxd-12 and Hoxd-13. These latter genes are members of the Hoxd complex and are expressed within the distal wrist (Hoxd 12) and within the hand and fingers (Hoxd 12 and 13). The role of the Hoxd 13 gene in the proximodistal differentiation of limb segments has been illustrated by the demonstration that mutations in the human gene transforms the metacarpals to carpals and metatarsals to tarsals. Likewise, overexpression of Hoxd13 in chick limb bud resulted in the transcriptional repression in the proximal part of the limb of Meis, the vertebrate ortholog of an homeo-box containing gene in drosophila called

homothorax (hth) that is required for proximal leg development

BMP antagonists in signaling networks. (a) Gremlin in limb-bud development. The predicted interactions between Gremlin, BMP4, FGF4 and Shh : Gremlin maintains FGF/Shh positive-feedback signaling during limb outgrowth by preventing BMP4 inhibition of this loop. Maintenance of this loop is essential for correct limb-bud development.

skeleton of the limb

The skeleton of the limb arises from somatic mesoderm by means of endochondral ossification. Formation of the intermediate segment (forearm) involves programmed apoptosis to separate a single mesenchymal condensation into two cartilage models (one for the radius and one for the ulna). In addition, separation of the digits depends on apoptosis within the interdigital grooves. Cartilage breaks down to form the joints in specific points. Periosteum, ligaments, tendons, and intramuscular connective tissues form from the noncondensed mesenchyme.

Skeleton of limb
In addition to somatic mesoderm, there are cells that migrate into the limb bud from the body wall. These cells are identified into three groups: (1) somitic components (somitic myotomes in particular) that migrate into the limb buds and give rise to all of the musculature of the limb (2) spinal nerves from the brachial plexus that go to the upper limb and from the lumbosacral plexus that go to the lower limb, and (3) blood vessel precursors going into the limb to provide the vasculature. By the end of the eighth week, the limb is perfectly formed. From there on out, the only remaining development is growth that is synchronized with that of the fetal body.

Skeleton formation
The skeletal elements of the limb develop from a column-like mesodermal condensation that appears along the long axis of the limb bud during the fifth week of gestation in human. With the exception of clavicle, the bones of the limbs form by ossification of a cartilaginous precursor according to a process called endochondral ossification.

Skeleton formation
Mesenchymal cells from the lateral plate condense to form prechondrogenic elements in the proximal region of the limb, giving rise to the anlagen of the humerus (or femur). Distal extension of the process results in the formation of the ulna and radius (or fibula and tibia) which further branches and segments to form the posterior proximal carpal (tarsal) element as well as the digital rays of digits IV-II. Prechondrocytes in the prechondrogenic condensations differentiate into chondrocytes in response to growth factors and secrete molecules characteristic of the extracellular matrix such as collagen type II and aggrecan (a large proteoglycan). The initial phase of chondrification results in the formation of a cartilaginous envelope, the perichondrium. This perichondrium in which bone morphogenetic protein 2, 4 and 7 (BMP 2, 4 and 7) and parathyroid hormone/ parathyroid hormone-related peptide receptor (PTH/PTHrPR) are expressed, inhibits chondrocyte proliferation and maturation thereby helping to control the growth and differentiation of the forming cartilage elements. As the cartilage elements grow different zones can be distinguished that demarcate the progressive differentiation of the chondrocytes. Cells at the ends of the elements are immature and undergo rapid proliferation.

Skeleton formation
Adjacent to the proliferation zone are the larger and more sparsely distributed prehypertrophic chondrocytes that express Indian hedgehog (Ihh), PTH/PTHrPR, BMP 6 and BMP receptor IA (BMPRIA). The terminally differentiated hypertrophic cells express a unique form of collagen, type X collagen, and eventually undergo programmed cell death and are replaced by osteoblasts. Defective cartilage growth occurs in a wide spectrum of disorders called chondrodysplasias that usually result in dwarfisms of variable severity. The most common of these disorders is achondroplasia , a dominant genetic disease caused by a recurrent activating mutation in the transmembrane domain of FGFR3 affecting chondrocyte proliferation and differentiation. The process of bone ossification begins in a region called the primary ossification center. Mesenchymal cells in the perichondrium differentiate into osteoblasts that secrete the calcium salt matrix of mineralized bone and form a primary bone collar around the bone which thickens as osteoblasts differentiate. In addition to chondrocytes and osteoblasts, a third cell type of hematopoietic origin, the osteoclasts contribute to skeletal remodeling throughout development. Indeed, the function of osteoblasts and osteoclasts is intimately linked since osteoblast synthesize and secrete molecules that control osteoclast differentiation.

(C) Schematic showing the contribution of the neural crest, lateral plate mesoderm, paraxial mesoderm, and notochord to the three major parts of the skeleton. (D) Mid-sagittal sections through the notochord of mouse embryos at the gestation days 12.5 (E12.5, top) and E15.5 (bottom). The E12.5 notochord is a rod-like structure that becomes surrounded by the mesenchymal cell condensations of the prospective vertebral bodies (VB) and IVD. E15.5 VB are cartilaginous and notochord cells have migrated into the intervertebral disc spaces, where they have formed NP. Sections are stained with nuclear fast red and with Alcian blue, which is specific of the notochord and cartilage extracellular matrix.

Fate and molecular control of skeletogenic mesenchymal cells.

hondrocyte early differentiation and development of cartilage primordia. (A) Alcian blue staining of a mouse embryo at E14.5 demonstrates that chondrocyte differentiation of skeletogenic cells leads to the formation of a primary skeleton that is entirely cartilaginous. (B) Sections through the developing paws of mouse embryos illustrate the major steps of early chondrogenesis. At E10.5, the limb bud is filled with skeletogenic cells. By E12.5, some of these cells have formed precartilaginous condensations that prefigure the future digits. By E14.5, condensed prechondrocytes have undergone chondrocyte early differentiation.

Chondrocyte maturation and development of cartilage growth plates.

(A) Sections through a mouse embryo tibia (T) illustrate the development of growth plates and endochondral bone. At E13.5, early chondrocytes in the center of cartilage primordia undergo prehypertrophic and hypertrophic maturation. They reach terminal maturation and are replaced by endochondral bone by E15.5. Later on, growth plates maintain themselves and elongate developing bones. Chondrocytes keep proliferating and give rise, layer by layer, to maturing chondrocytes. These cells, which eventually die, are replaced by bone. The sections are stained with Alcian blue and nuclear fast red. (B) Schematic of the molecular control of GP chondrocytes.

Osteoblast differentiation and intramembranous and endochondral ossification.

(A) Sections through an endochondral bone in a newborn mouse show the replacement of cartilage by bone. The left section is stained with Alcian blue and the right one with the von Kossa reagent, which leaves a brown precipitate on the mineralized bone matrix. (B) Schematic showing how GP chondrocytes and boneforming cells interact with each other to achieve endochondral ossification. (C) Coronal sections of a newborn mouse head. In the suture linking the two frontal bones (top panel), osteoblast precursors are surrounded by an abundant collagenous matrix. Further away (bottom panel), osteoblasts mature and deposit a mineralized bone matrix. This matrix is stained with the von Kossa reagent. (D) Schematic of the molecular control of osteoblast differentiation.

Synovial joint development.

(A) Sections through the mouse knee joint at various stages of development. At E12, the presumptive joint region (arrow) is not distinguishable from the femur (F) and T precartilaginous condensations. At E13.5, this region becomes distinguishable as surrounding cartilage primordia are overtly developing. At E16.5, joint morphogenesis is well advanced. The joint cavity has formed between the patella (P) and F. Cruciate ligaments and FP, lined with synovial tissue, are developed. At the postnatal day 19, the knee joint is mature. The AC is separated from the epiphyseal GP by a secondary center of ossification. The sections are stained with Alcian blue and nuclear fast red. (B) Schematic of the molecular control of synovial joint cell differentiation.

Models for the development of sexually dimorphic digit proportions AR (blue circles) and ER (pink circles) are present in the digit condensations of male and female embryos, with higher levels found in 4D . (A) In males, digits are exposed to high levels of circulating androgen and low levels of circulating estrogen, which results in preferential binding and activation of AR (ARA represents the androgen bound to the AR). High AR activity and low ER activity (ARA/er) in males leads to differential gene expression profiles in 4D relative to 2D (green indicates genes higher in 4D, and red indicates genes higher in 2D). In turn, chondrocyte proliferation is increased in the proximal phalanx of 4D, which results in elongation of 4D relative to 2D, leading to a lower 2D:4D ratio. (B) In females, digits are exposed to high levels of estrogen and low levels of androgen, leading to preferential binding and activation of ER (ERE). Low AR activity and high ER activity (ar/ ERE) induces an opposite shift in the skeletogenic gene expression profile of 4D relative to 2D (indicated by gene names in green and red, as above). Higher levels of activated ER cause decreased chondrocyte proliferation in the middle phalanx of 4D, which reduces its growth relative to 2D and results in a higher 2D:4D ratio.

Summary molecular control

Limb Initiation FGF FGF10 , FGF8 (lateral plate intermediate mesoderm) prior to bud formation FGF8 (limb ectoderm) FGFR2 FGF can respecify Hox gene expression (Hox9- limb position) Hox could activate FGF expression Limb Specification (Fore- Hind-) regulated by T-box genes (transcription factor) Tbx5- forelimb Tbx4 leg Limb Axes Limb Patterning- Axes Signals give positional information which is interpreted by Hox gene expression establishing programs of differentiation. Proximodistal Axis Dorsoventral Axis Anteroposterior Axis

Summary

Limb Patterning- Axes :Wing has been used as Model of limb development as chick wing easy to manipulate: removal, grafting, additional ARER, ZPA etc Limb Patterning- Axes Proximodistal Axis AER formed by Wnt7a then AER secretes FGF2, 4, 8 stimulates proliferation and outgrowth Dorsoventral Axis somite provides dorsal signal to mesenchyme which dorsalizes ectoderm ectoderm then signals back (Wnt7a) to mesenchyme to pattern limb Anteroposterior Axis ZPA zone of polarizing activity mesenchymal posterior region of limb addition of extra ZPA duplicated digits signal is Shh